Therapeutic agents containing cannabis flavonoid derivatives targeting kinases, sirtuins and oncogenic agents for the treatment of cancers

ABSTRACT

A  cannabis -based flavonoid pharmaceutical composition including any one or more selected from among the group of Cannflavin A, Cannflavin B. Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside and their derivatives selected from among the group of Geraldol, Rhamnetin, Isorhamnetin, Rhamnazin, or their synthases, for the prevention and treatment of certain cancers that can be treated by therapeutically targeting oncogenic factors including kinases, sirtuins, bromodomains, matrix metalloproteinases and histone acetylases. Some of the cancers that can be treated by use of  cannabis  flavonoids based on the inhibition of these therapeutic targets include but are not limited to brain, breast, colon, renal liver, lung, pancreatic, pigmented villonodular synovitis, prostate, leukemia, melanomam, tenosynovial giant cell tumor, as well as any other cancers that overexpress the oncogenic factors inhibited, by the  cannabis  flavonoids or their derivatives herein identified.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patent application Ser. No. 14/835,198 filed 25 Aug. 2015, and is a continuation-in-part of PCT Patent Application PCT/US15/62331 filed 24, Nov. 2015.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to flavonoid derivatives and, more particularly, to cannabis flavonoid derivatives or the pharmaceutically acceptable salt thereof that may be used in a pharmaceutical composition for preventing and treating cancer.

2. Description of the Background

Flavonoids are common constituents of plants and cover a wide range of functions including acting as yellow pigments in petals and leaves to attract pollinating insects. They might also appear as bluish pigments (anthocyanins) to receive certain wavelengths of light, which permits the plant to be aware of the photoperiod. Many of these flavonoids also protect the plants by being involved in the filtering of harmful ultraviolet light. Some flavonoids play crucial roles in establishing symbiotic fungi, while at the same time they fight infections caused by pathogenic fungi.

Flavonoids have relevant pharmacological activities such as: antioxidant, antidiabetic, anti-inflammatory, antiallergic, antibiotic, antidiarrheal, CNS and against cancer.

Cannabis is credited to have several beneficial pharmacological properties. Unfortunately much attention on Cannabis is focused on its recreational use as a psychoactive drug. Studies have identified over twenty flavonoids in the Cannabis plant, such as: cannflavin A, cannflavin B, cannflavin C, chrysoeriol, cosmosiin, flavocannabiside, vitexin, isovitexin, apigenin, kaempferol, myricetin, quercetin, luteolin, homoorientin and orientin. Turner, C. E., Elsohly, M. A., & Boeren, E. G., “Constituents of Cannabis Sativa L. XVII, A review of the natural constituents”, Jowurnal of Natural Prodrccts, 43(2), 169-234 (1980). The distribution of these flavonoids in the plant varies depending on the type of flavonoid. The total content of flavonoids in the Cannabis' leaves and flowers can reach 1-2.5% of its dry weight depending on environment factors and the variety of the plant. It is noteworthy to mention that even though cannflavin A has been isolated from other plant sources, it is only cannabis that has been shown to harbor all three natural cannflavins.

Cannabis flavonoids have been shown to have several pharmacological properties especially the most common flavonoids such as quercetin, apigenin, luteolin and kaempferol. ElSohly, M. A., Slade, D., “Life Sciences”, 78(5), 539-548 (2005). Chemical constituents of marijuana: the complex mixture of natural cannabinoids). These more common flavonoids can be found in many other plants and as such are not unique to cannabis. Apart from the specific pharmacologic properties identified, cannabis flavonoids are thought to play synergistic roles with other metabolites in the plant. For example, some flavonoids are volatile, lipophilic, permeate membranes, and seem to retain pharmacological properties in cannabis smoke. Sauer, M. A., Rifka, S. M., Hawks, R. L, Cutler, G. B., & Loriaux, D. L, “Journal of Pharmacology and Experimental Therapeutics”, 224(2), 404-407 (1983). Marijuana: interaction with the estrogen receptor. Flavonoids may modulate the pharmacokinetics of THC, via a mechanism shared by CBD, the inhibition of P450 3A11 and P450 3A4 enzymes. These two related enzymes metabolize environmental toxins from procarcinogens to their activated forms. P450-suppressing compounds as such serve as chemoprotective agents, shielding healthy cells from the activation of benzo atpyrene and aflatoxin BI. Offord, E. A., Mace, K., Avanti, O., & Pfeifer, A. M., “Mechanisms Involved In The Chemoprotective Effects Of Rosemary Extract Studied In Human Liver And Bronchial Cells”, Cancer Letters, 114(1), 275-281, (1997). Benzo[α]pyrene and aflatoxin BI are two procarcinogenic agents found in cannabis smoke. McPartland, J. M., & Pruitt, P. L., “Alernative Therapies In Health And Medicine”, 5(4), 57 (1999). Side effects of pharmaceuticals not elicited by comparable herbal medicines: the case of tetrahydrocannabinol and marijuana. Cannabis flavonoids thus may be modulating the therapeutic effects of THC and CBDs by either synergistically enhancing desired pharmnnacologic effects or reducing detrimental effects. McPartland, J. M., Russo, E. B., “Cannabis And Cannabis Extracts: Greater Than The Sum Of Their Parts?”, Journal of Cannabis Therapeutics, 1(3-4), 103-132 (2001).

There is a small amount of literature on the bioactivity of cannflavins and other closely related flavonoids isolated either from cannabis or from other plants. Banett et al (1985) reported the inhibition properties of cannflavins on prostalglandins with implication on inflammation. Barrett, M. L., Gordon, D., Evans, F. J., “Isolation From Cannabis Sativa L. Of Cannflavin—A Novel Inhibitor Of Prostaglandin Production”, Biochemical Pharmacology, 34(11), 2019-2024 (1985).

Blanco et al. (2008) reported on cannabidiol and denbinobin and their use for the prevention and treatment of gastrointestinal inflammatory diseases and for the prevention and treatment of gastrointestinal cancers. U.S. patent application Ser. No. 12/681,453 published 2 Sep. 2010. Radwan et al. (2008) reported antileishmanial activity for cannflavin A and canaflavin B. Radwan, M. M., ElSohly, M. A., Slade, D., Ahmed, S. A., Wilson, L., El-Alfy, A. T., Ross, S. A., “Non-Cannabinoid Constituent From A High Potency Cannabis Sativa Variety”, Phytochemistry 69(14), 2627-2633 (2008). Brunelli, et al. (2009) reported that isocanniflavin B induced autophagy in hormone sensitive breast cancer cells. Brunelli, E., Pinton, G., Bellini, P., Minassi, A., Appendino, G., & Moro, L, “Flavinoid-Induced Autophagy In Hormone Sensitive Breast Cancer Cells”, Fitoterapia 80(6), 327-332 (2009). Li and Meng (2012) reported the use of the flavonoid Icaritin to treat estrogen receptor related disease. U.S. Pat. No. 8,252,835 issued 28 Aug. 2012. Meng et al. (2014) also reported the use of Icaritin to treat cancers. U.S. patent application Ser. No. 14/291,639 published 24 Dec. 2014. Apart from the autophagy activity on breast cancer reported by Brunelli and colleagues no other report was seen relating to anticancer activity of cannflavins. Cytotoxicity studies carried out by the US National Cancer Institute (NCI) using its 60 cancer cell line panel showed that cannflavin B was not cytotoxic against cancer cells (NSC:719330).

Apart from the Cannflavins, a number of other flavonoid derivatives in Cannabis are known to exhibit varying anti-cancer properties. The most prominent of these flavonoids with reported anticancer activity include apigenin, see Shukla, Sanjeev, and Sanjay Gupta, “Apigenin: a promising molecule for cancer prevention”, Pharmaceutical research 27.6 (2010): 962-978; Chrysin: Khoo, Boon Yin, Siang Ling Chua, and Prabha Balaram. “Apoptotic effects of chrysin in human cancer cell lines”, International journal of molecular science, 11.5 (2010): 2188-2199. Chrysoeriol: Yang, Yang, et al. “Discovery of chrysoeriol, a PI3K-AKT-mTOR pathway inhibitor with potent antitumor activity against human multiple myeloma cells in vitro”, Journal of Huazhong University of Science and Technology [Medical Science] 30 (2010): 734-740; Luteolin: Chowdhury, Arnab Roy, et al., “Luteolin, An Emerging Anti-Cancer Flavonoid, Poisons Eukaryotic DNA To Poisomerase 1.”, Biochemical Journal 366.2 (2002): 653-661; and Quercetin: Chen, Jie, et al. “Combination with water-soluble antioxidants increases the anticancer activity of quercetin in human leukemia cells”, Die Pharmazie—An International Journal of Pharmaceutical Sciences 59.11 (2004): 859-863.

Despite the foregoing, there has been little prior effort to detail the use of flavonoid derivatives for a “molecular treatment approach”, e.g., the targeting of kinases, sirtuins, matrix metalloproteinase, bromodomains and histone deacetylases by the cannflavins, cannabis flavonoids or their analogs in the treatment of cancer utilizing FDA approved therapeutic targets. An example of an FDA recognized therapeutic target is FLT3 internal tandem duplications (FLT3-ITDs) which are present in nearly 25% of patients with AML and have been associated with poor response to conventional chemotherapy. See, Abu-Duhier, F. M., et al., “FLT3 Internal Tandem Duplication Mutations In Adult Acute Myeloid Leukaemia Define A High-Risk Group”, British journal of haematology 111.1 (2000), 190-195: Wiemik, Peter H., “FLT3 inhibitors for the treatment of acute myeloid leukemia”, Clin Adv Hematol Oncol 8.6 (2010): 429-444. Chin and colleagues attempted a study of 17 flavonoids for their inhibition of FLT3 kinase but none of the flavonoids showed activity at a therapeutically relevant dose. See Chin, Young-Won, Jae Yang Kong, and Sun-Young Han, “Flavonoids as receptor tyrosine kinase FLT3 inhibitors”, Bioorganic & medicinal chemistry letters 23.6 (2013): 1768-1770. Meng and colleagues reported the activity of the flavonoid Icaritin against FLT3 and the acute myeloid leukemia cell line. See, PCT Patent Application WO/2015/024512 (2015). The first FDA approved drug for the treatment of acute myeloid leukemia harboring the FLT3-ITD mutation is Midostaurin. See, DiGiulio, Sarah. “FDA's Breakthrough Therapy Designation to PKC412 (Midostaurin) for AML”, Oncology Times (2016). Other drugs targeting this very important kinase and currently in advance phases of clinical development include Quizartinib (see, Carlson, Robert H. “AML: FLT3 Inhibitor Quizartinib Produces High Response Rates in Relapsed/Refractory Patients”, Oncology Times 35.6 (2013): 14-15, also, Gilteritinib, Pratz, Keith W., and Mark Levis, “How I treat FLT3-mutated AML”, Blood (2016): blood-2016. FLT3 and a number of other kinases such as the CSFIR which is potently inhibited by FBL-03G are considered to be oncogenic factors. CSFI R is another attractive therapeutic target involving a number of cancers. Ravi, Vinod, Wei-Lien Wang, and Valerae O. Lewis. “Treatment of tenosynovial giant cell tumor and pigmented villonodular synovitis.” Current opinion in oncology 23.4 (2011): 361-366. Strachan, Debbie C., et al. “CSFI R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8+ T cells.” Oncoimmunology 2.12 (2013): e26968. Xu, Jingying, et al. “CSFIR signaling blockade stanches tumor-infiltrating myeloid cells and improves the efficacy of radiotherapy in prostate cancer.” Cancer research 73.9 (2013): 2782-2794.

An oncogene is a gene that has the potential to cause or promote the progression of cancer. In tumor cells, oncogenes are often mutated or expressed at abnormal levels. As seen in the example of FLT3, some cancers overexpress certain oncogenic factors, and these oncogenic factors may be useful therapeutic targets. However, different cancers respond differently to various oncogenic agents and that is why the response of an outgrowth line to a combination of several agents is unpredicatble. The absence of knowledge regarding the use of canflavins and cannabis flavonoids or their derivatives against these important therapeutic targets has been problematic, but prompted research by the present inventors on cannflavins and their analogs that has led to the present invention: a novel small natural cannabis-based flavonoid molecule that is a first-in-class selective FLT3 and FLT3-TD inhibitor with no detectable toxicity or safety screen issues. The molecule exhibits significant in-vivo efficacy against several kinases, sirtuins, matrix metalloproteinases, bromodomains and histone deacetylases, with excellent results.

SUMMARY OF THE INVENTION

It is another object to provide a method for the prevention and treatment of cancer using specific cannabis-based flavonoid pharmaceutical compositions.

It is another object to provide a method for isolating specific cannabis-based flavonoid pharmaceutical compositions from raw plant material that are biologically active in the prevention and treatment of cancer.

It is still another object to provide a method for synthesizing said specific cannabis-based flavonoid pharmaceutical compositions.

In accordance with the foregoing objects, the inventors have isolated and successfully synthesized cannflavins including cannflavin A, cannflavin B and cannflavin C and their derivative flavonoids including chrysoeriol, diosmetin, geraldol and their derivatives thereof and have demonstrated their anticancer efficacy in various assays including with specific focus on identifying their molecular therapeutic targets. The present invention relates to the use of the newly synthesized flavonoids alone or in combination with other bioactive compounds to treat or prevent cancers.

In accordance with the foregoing objects, the present invention provides a flavonoid-based pharmaceutical composition for the prevention and treatment of cancer having the structure of the general formula shown below (see also FIG. 1) or a pharmaceutically acceptable salt thereof.

wherein,

R1-R10 may be any one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), a methyl group comprising one carbon atom bonded to three hydrogen atoms (CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl), Bromine (Br), Fluorine (F), Glutamic acid (Glu), geranyl chain, prenyl chain and any salts or derivatives of the foregoing. A and B may each be either a single or double bond.

A method for the prevention and treatment of cancer using the specific cannabis-based flavonoid pharmaceutical compositions is also disclosed, as well as a method for isolating the specific flavonoid-based pharmaceutical compositions from raw plant material, and a method for synthesizing said flavonoid-based pharmaceutical compositions.

The present invention is described in greater detail in the detailed description of the invention, and the appended drawings. Additional features and advantages of the invention will be set forth in the description that follows, will be apparent from the description, or may be learned by practicing the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, features, and advantages of the present invention will become more apparent from the following detailed description of the preferred embodiments and certain modifications thereof when taken together with the accompanying drawings in which:

FIG. 1A is an illustration of the general cannabis-based flavonoid pharmaceutical compositions according to the present invention.

FIG. 1B is an illustration of the specific family of cannabis-based flavonoid pharmaceutical compositions best-suited to be therapeutic targets for the cancer AML.

FIG. 2 is a flow diagram illustrating a suitable method for isolating the specific cannabis-based flavonoid pharmaceutical compositions from raw plant material.

FIG. 3 is a process diagram illustrating a suitable synthesis approach.

FIG. 4 is an illustration of the specific isolated cannabis-based flavonoid pharmaceutical compositions including Flavone, Flavanone and Flavanol isolates and their synthetic derivatives, according to the present invention.

FIG. 5 is a graphical illustration of the results of the kinase inhibition by cannabis flavonoids and derivatives presented in Table 1 and Table 4

FIG. 6 is a graphical illustration of the results of the anticancer activity presented in Table 3 (Hela cells at A and CMK cells at B) and Table 4 (MV4-11, MOLT14 and AML29).

FIG. 7 is a graphical illustration of the results of the anticancer activity in mice bearing pancreatic cancer treated with FBL-03B.

FIG. 8 is a graphical illustration of the results of the anticancer activity in mice bearing acute myeloid leukemia harboring FLT3 mutation.

FIG. 9 is a graphical illustration of the results of the anticancer activity in mice bearing pancreatic cancer treated with FBL-03G FIG. 10 presents the mechanism of inhibition of FLT3 and FLT3-ITD by FBLGS-70.

FIG. 11 presents the computer aided drug design study of FBLGS-70 and FBLGS-81 docking interaction with the crystal structure of FLT3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts.

An oncogene is herein defined as a gene that has the potential to cause cancer. In tumor cells, oncogenes are often mutated or expressed at high levels. Certain cancers overexpress certain oncogenic factors including kinases, sirtuins, bromodomains, matrix metalloproteinases and histone deacetylases. Thus, these particular oncogenic factors are identified as useful therapeutic targets for purposes of the present invention. Given the foregoing targets, some of the cancers that can be treated by use of cannabis flavonoids based on the inhibition of these therapeutic targets include brain, breast, colon, renal, liver, lung, lymphoma, pancreatic, pigmented villonodular synovitis, prostate, leukemia, melanoma, tenosynovial giant cell tumor as well as any other cancers (solid, soft or heamatological) that overexpress the oncogenic factors inhibited by the identified cannabis based flavonoids.

The present invention is a group of cannabis-based flavonoid pharmaceutical compositions selected from among the group of Apigenin, Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside, Kaempferol, Luteolin, Myricetin, Orientin, Isoorientin (Homoorientin), Quercetin, (+)-Taxifolin, Vitexin, and Isovitexin and their derivatives including geraldol, rhamnetin, isorhamnetin, rhamnazin, useful for the prevention and treatment of certain cancers by targeting kinases, sirtuins, bromodomains, matrix metalloproteinases and histone deacetylases which have been identified to be useful therapeutic targets for said cancers.

Some of the cancers that can be treated by use of cannabis flavonoids based on the inhibition of these therapeutic targets include brain, breast, colon, renal, liver, lung, lymphoma, pancreatic, pigmented villonodular synovitis, prostate, leukemia, melanoma tenosynovial giant cell tumor as well as any other cancers (solid, soft or heamatological) that overexpress the oncogenic factors inhibited by the cannabis flavonoids identified under this invention.

The cannabis-based flavonoid pharmaceutical composition for the prevention and treatment of cancers has the structure of the general formula shown below (see also FIG. 1), or a pharmaceutically acceptable salt thereof.

wherein,

R1-R10 may be any one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), a methyl group comprising one carbon atom bonded to three hydrogen atoms (CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl), Bromine (Br), Fluorine (F), Glutamic acid (Glu), and any salts or derivatives of the foregoing. A and B may each be either a single or double bond.

As described below the present inventors have tested candidates from the general grouping on various cancer cell lines and were further able to identify the most potent family of candidates as therapeutic targets for the particular cancer AML, the family being selective FLT3, FLT3-TD and FLT-3 D835Y inhibitors with low toxicity and safety screen issues during in vitro profiling. This family of AML-therapeutic targets likewise has the flavone backbone (2-phenyl-1,4-benzopyrone) chemical structure, in this case more defined as shown below (see also FIG. 1B), or any pharmaceutically acceptable salt thereof:

R2 may be one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), or an oxygen molecule (O);

R3 may be none, or one or more hydrogen molecules (H),

R4 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or a nitrogen dioxide molecule (NO2);

R5 and R8 may be none, or a hydroxide molecule (OH);

R7 may be none, or the amine NH2;

R9 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or an oxygen molecule (O); and A and B may each be either a single or double bond.

From the family of AML-therapeutic targets the inventors isolated one particular flavonoid molecule (FBLGS-70 described below) that proved to be a first-in-class selective FLT3, FLT3-ITD and FLT-3 D835Y inhibitor with no detectable toxicity or safety screen issues. The molecule FBLGS-70 in particular exhibited significant in-vivo efficacy on MV4-11 AML. Thus, FBLGS-70 and its derivatives were synthesized, and structure activity relationship studies were conducted. From these the inventors identified FBLGS-81, an isomer of FBLGS-70 also described below, as the most potent candidate to advance for further development against AML.

FIG. 2 is a flow diagram illustrating a suitable method for isolating the specific cannabis-based flavonoid pharmaceutical compositions from raw plant material.

Thus, from the general family, the most preferred flavonoid molecules for use in combating AML are primarily FBLGS-81 and secondarily FBLGS-70, both Type I selective tyrosine kinase inhibitor with potent activity against FLT3, FLT3-ITD and FLT3-D835Y kinases.

FIG. 3 shows a molecular illustration of the specific isolated cannabis-based flavonoid pharmaceutical compositions of the present invention including Flavone, Flavanone and Flavanol isolates and their synthetic derivatives, complete with a process diagram illustrating a suitable synthesis approach for each.

A method for the prevention and treatment of cancer using the specific cannabis-based flavonoid pharmaceutical compositions above is also disclosed. Administration may be by various routes including oral, rectal or intravenous, epidural muscle, subcutaneous, intrauterine, or blood vessels in the brain (intracerebroventricular) injections. The flavonoid derivatives of the general formula (FIG. 1) according to the present invention and a pharmaceutically acceptable salt thereof may be administered in an effective dose, depending on the patient's condition and body weight, extent of disease, drug form, route of administration, and duration, within a range of from 0.1 to 500 mg between 1-6 times a day. Of course, most dosages will be by a carrier. The specific dose level and carrier for patients can be changed according to the patient's weight, age, gender, health status, diet, time of administration, method of administration, rate of excretion, and the severity of disease.

The composition may be formulated for external topical application, oral dosage such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, suppositories, or in the form of a sterile injectable solution. Acceptable carriers and excipients may comprise lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl benzoate, propyl benzoate, talc, magnesium stearate, and mineral oil.

Bioactivity

Bioactivity of the above-described compounds were verified and is presented in Tables 1, 2, 3 and 4 below:

TABLE 1 FBL-03A FBL-03B FBL-03C FBL-03D FBL-03G Kinase IC₅₀ (nM) Aurora A 730 12 >30000 1090 BIKE >20000 >20000 21 >30000 >20000 CK2a 740 768 58 >30000 38 CK2a2 350 477 19 >30000 9.7 c-Kit(Y823D) >20000 244 >20000 >30000 84 c-Kit(D820Y) >20000 1280 >20000 >30000 113 DRAK2 >20000 <1000 980 >30000 >10000 DYRK1/DYRK1A >20000 <1000 620 >30000 36 DYRK1B >20000 1670 6400 >30000 22.8 EFGR(L858R, >20000 >1000 680 >30000 >1000 T7790M) EPHB6 >20000 >1000 270 >30000 >1000 FGR >20000 224 >20000 >30000 880 FLT3 >20000 41.9 >20000 >30000 44 FLT3(D835Y) >20000 12.7 >20000 >30000 45 FLT3(D835V) 220 <1000 190 >30000 <1000 FLT3(ITD) >20000 57 >20000 >30000 <1000 FLT4(VEGFR3) 330 9.3 >20000 >30000 4220 FMS/CSF1R 1500 199 1200 >30000 4 JAK3 >20000 >1000 780 >30000 >20000 KIT 350 <1000 >1000 >30000 <1000 KIT(L576P) 180 <1000 >1000 >30000 <1000 KIT(V559D) 200 <1000 >1000 >30000 <1000 MELK >20000 232 >1000 >30000 105 MEK5 140 >1000 84 >30000 >1000 PASK >20000 2060 >20000 >30000 116 PDGFRa — 982 — >30000 5920 PDGFRa(T674I) — 0.92 >1000 >30000 2360 PDGFRB 330 1160 >1000 >30000 3310 PIK3CA(1800L) >20000 780 >30000 >20000 PIK4CB 670 >1000 >30000 >20000 PIM-1 >20000 >1000 >30000 78 PIM-3 >20000 173 >1000 >30000 35 PIP5K1A >20000 >10000 360 >30000 >20000 RIOK1 >20000 >10000 340 >30000 >20000

TABLE 2 Cell Line FBL-03A FBL-03B FBL-03C FBL-03D FBL-03G RIOK3 >20000 >10000 280 >30000 >20000 SIK2 >20000 >10000 >1000 >30000 63 SRPK1 >20000 >10000 300 >30000 >10000 TNIK >20000 152 >1000 >30000 115

TABLE 3 Isolation And Synthesis Activity FBL-03A FBL-03B FBL-03C FBL-03D FBL-03G SIRT IC₅₀ (μM) SIRT-1 19.00 27.40 39.50 — — SIRT-2 2.57 10.80 14.00 2.38 24.10  SIRT-3 94.90 77.00 65.40 — 66.40  SIRT-5 523.00 104.00 132.00 — 974.00  Bromodomain IC₅₀ (μM) BRD2 NT 9.52 NT NT 12.00  BRD3 NT 7.05 NT NT 8.69 BRD4 NT 10.40 NT NT 6.14 Matrix metalloproteinase IC₅₀ (μM) MMP-2 NT 115.00 NT NT 6.64 MMP-3 NT — NT NT 66.30  MMP-7 NT 17.52 NT NT 3.35 MMP-9 NT — NT NT 85.40  IC₅₀ (μM) BCL-2 NT — NT NT 2.49 BCL-XL NT — NT NT — IC₅₀ (μM) A498 (Kidney) 17 NT 14 NT NT A549 (Lung) 17 NT 9.4 NT NT CFPAC-1 (Pancreatic) 12 17 12 NT 14.32  CMK (leukemia) NT 11.60 NT NT 1.78 COLO-205 (Colon) 27 NT 17 NT NT DLD-1 (Colon) 15 NT 13 NT NT HC-1 (Leukemia) NT 29.70 NT NT 5.00 HeLa (cervical) NT 10.40 NT NT 2.53 IGROV-1 (Ovarian) 29 15 NT NT KMS-11 (Multiple NT NT NT NT NT myeloma) MCF-7 (Breast) 17 NT 12 NT NT MiaPaca-2 16 NT 9.5 NT NT (Pancreatic) MOLT-4 (Leukemia) NT 13.20 NT NT 20.00  MV4-11 (Leukemia) NT 1.43 NT NT 3.1  NCI-H69 (Small lung) 16 11 18 NT 9.5  PC-3 (Prostate) 26 NT 20 NT NT RL (Lymphoma) 5.9 NT 12 NT NT SNU-16 (Stomach) NT 15.00 NT NT 4.09 U2-OS (Bone) NT 19.40 NT NT 8.70 UACC-62 27 NT 14 NT NT (Melanoma) U87 (Glioma) 34.00 6.20 12.50 NT 5.46

A method for isolating the specific cannabis-based flavonoid pharmaceutical compositions from raw plant material is also disclosed. The isolation was realized according to the scheme shown in FIG. 2.

At step 10 an appropriate amount of plant biomass is collected. For present purposes, Cannabis sativa plants were collected by hand. See, Radwan, M. M., ElSohly, M. A., Slade, D., Ahmed, S. A., Wilson, L., EI-Alfy, A T., Khan, I A., Ross, S. A., “Non-Cannabinoid Constutuents From A High Potency Cannabis Sativa Variety”, Phytochemistry 69, 2627-2633 (2008) and Radwan, M. M., Ross, S. A., Slade, D., Ahmed, S. A., Zulftiqar, F., ElSohly, M. A., “Isolation And Characterization Of New Cannabis Constituents From A High Potency Variety”, Planta Med. 74, 267-272 (2008). The collected plant material was air dried under shade and pulverized into powder.

At step 20 the powder is subjected to supercritical fluid extraction (SFE) by which carbon dioxide (CO²) is used for separating one component (the extractant) from another (the matrix). The extract is evaporated to dryness resulting in a green residue.

At step 30, for experimental purposes, a bioassay-guided fractionation was employed, using a standard protocol to isolate a pure chemical agent from its natural origin. This entailed a step-by-step separation of extracted components based on differences in their physicochemical properties, and assessing all their biological activity. The extracted components may, for example, be fractionated by dry column flash chromatography on Si gel using hexane/CH₂Cl₂/ethyl acetate and mixtures of increasing polarity to yield different fractions. The sample is then degassed by ultra-sonication to yield an insoluble solid, which solid is then filtered. The sample may then be subjected to high performance liquid chromatography (HPLC) using a column Phenomenex Luna™ C18, 5 μm, 2×50 mm; eluent, acetonitrile with 0.05% MeOH to confirm the presence of the various fractions.

At step 40, bioactivity of the extracts were verified by an anticancer cell proliferation assay as described above. This identified the bioactive flavonoids from all the supercritical fluid extracts (SEE). As reported previously, the identified cannabis-based flavonoid extracts showed activity against several cancer cell lines including brain, breast, Kaposi sarcoma, leukemia, lung, melanoma, tenosynovial giant cell tumor ovarian, pancreatic, colon and prostate cancer.

At step 50 Nuclear Magnetic Resonance Spectroscopy and mass spectrometry (NMR/MS) was performed and the interpreted spectra were consistent with cannabis-based flavonoid compositions as identified above, as illustrated in step 60.

Synthesis

Given the known structure of the general formula of FIG. 1 and the isolate of FIG. 2, a method for synthesizing the same becomes possible. The bioactive cannabis-based flavonoid pharmaceutical composition may be synthesized by the phenylpropanoid metabolic pathway in which the amino acid phenylalanine is used to produce 4-coumaroyl-CoA.

FIG. 3 is a process diagram illustrating a suitable synthesis approach for the cannflavins. The 2′,4′,6′-Trihydroxyacetophenone was the major starting material and the synthesis was carried out using art known to the industry with modifications yielded the flavonoid backbone, which contains two phenyl rings for the cannflavins. Conjugate ring-closure of chalcones results in the familiar form of flavonoids, the three-ringed structure of a flavone. The metabolic pathway continues through a series of enzymatic modifications to yield the desired Flavone, Flavone and Flavanol as identified above and as shown in step 60 (FIG. 3). The specific Flavone, Flavanone and Flavanol isolates are shown in step FIG. 4.

For background see Minassi, A., Giana, A., Ech-Chahad, A., & Appendino, G. “A regiodivergent synthesis of ring A C-prenylflavones”. Organic Letters 10(11), 2267-2270 (2008). Of course, one skilled in the art will readily understand that other methods for synthesis are possible, such as the asymmetric methods set forth in Nibbs, A E, Scheidt, K A, “Asymmetric Methods for the Synthesis of Flavanones, Chromanones, and Azaflavanones”,

European Journal Of Organic Chemistry, 449-462. doi: 10.1002/ejoc.201101228. PMC 3412359, PMID 22876166 (2012). Borsari, Chiara, et al. “Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs.” Journal of Medicinal Chemistry 59.16 (2016): 7598-7616. Pandurangan, N. “A new synthesis for acacetin, chrysoeriol, diosmetin, tricin and other hydroxylated flavones by modified baker-venkataraman transformation.” Letters In Organic Chemistry 11.3 (2014): 225-229. Wang, Q. Synthesis of citrus bioactive polymethoxyflavonoids and flavonoid glucosides. Chinese journal of organic chemistry, 2010, 30, 11, 1682-1688.

Bioactivity Assays

Cannabis flavonoids and their analogs were subjected to kinase inhibition assay. The compounds were first screened at a single concentration of 10 μM in the primary assay. Compounds inhibiting at least 70% of specific kinases were subjected to further screening to determine kd/IC₅₀ values. To determine the kd or IC₅₀ values, competition binding assays were established, authenticated and executed as described previously. Fabian et al., “A Small Molecule-Kinase Interaction Map For Clinical Kinase Inhibitors.”, Nat Biotechnol, 23(3):329-36, Epub (2005). See also, Karaman et al., “A Quantitative Analysis Of Kinase Inhibitor Selectivity”, Nat. Biotechnol. January, 26(1): 127-32. doi: 10.1038/nbt1358 (2008). For most assays, kinases were fused to T7 phage strains (Fabian, supra) and for the other assays, kinases were produced in HEK-293 cells after which they were tagged with DNA for quantitative PCR detection. In general, full-length constructs were used for small, single domain kinases, and catalytic domain constructs for large multi-domain kinases. The binding assays utilized streptavidin-coated magnetic beads treated with biotinylated small molecule ligands for 30 minutes at room temperature which generated affinity resins for the kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 40× stocks in 100% DMSO and diluted directly into the assay (Final DMSO concentration=2.5%). All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by quantitative PCR. An illustration of the kinase interaction process is presented below. Kd/IC₅₀ values were determined using a standard dose response curve using the hill equation. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

Percent Control (% Ctrl)

The compound(s) were screened at 10 μM and results for primary screen binding interactions are reported as ‘% Ctrl’, where lower numbers indicate stronger hits in the matrix.

$\% \mspace{14mu} {Ctrl}\mspace{14mu} {{Calculation}\mspace{11mu} \left\lbrack \frac{{{test}\mspace{14mu} {compound}\mspace{14mu} {signal}} - {{positive}\mspace{14mu} {control}\mspace{14mu} {signal}}}{{{negative}\mspace{14mu} {control}\mspace{14mu} {signal}} - {{positive}\mspace{14mu} {control}\mspace{14mu} {signal}}} \right\rbrack} \times 100$

where: test compound=compound submitted by Environmental Health Foundation negative control=DMSO (100% Ctrl) positive control=control compound (0% Ctrl).

The results of the kinase inhibition by cannflavins and flavonoid derivatives are presented in Table 1 and Table 4 (below) and in FIG. 5 and FIG. 10.

TABLE 4 Acute Myeloid Leukemia Acute Myeloid Leukemia cell Normal Kinases lines Bone FLT3 FLT3-ITD FLT3-D835Y MV4-11 MOLM-14 THP-1 marrow Code IC50 (μM) FBLGS-70 0.011 0.006 0.021 1.25 1.7 >100 >100 FBLGS-73 0.127 0.079 0.022 3.26 3.68 >100 >100 FBLGS-73 0.152 0.046 0.048 4.53 4.86 >100 >100 FBLGS-74 16.6 9.09 8.58 >100 >100 >100 >100 FBLGS-75 0.220 0.091 0.076 4.35 4.57 >100 >100 FBLGS-76 0.87 0.75 0.68 1.9 2.3 >100 >100 FBLGS-77 1.32 0.799 0.577 7.21 7.34 >100 >100 FBLGS-78 0.307 0.345 0.149 5.62 5.73 >100 >100 FBLGS-79 0.025 0.084 0.013 3.41 4.84 >100 >100 FBLGS-80 0.060 0.084 0.027 5.86 6.12 >100 >100 FBLGS-81 0.0019 0.0038 0.0009 0.9 0.7 >100 >100 FBLGS-82 17.4 12.1 11.0 >10 >10 >100 >100 FBLGS-83 0.578 0.180 0.082 1.6 2.35 13.0 NT FBLGS-84 0.822 0.453 0.194 6.3 7.42 NA >100 FBLGS-85 0.050 0.057 0.022 0.9 1.5 >100 >100 FBLGS-86 0.074 0.045 0.043 2.2 3.1 >100 >100 Midostaurin 0.0004 0.001 0.00009 0.008 0.022 >100 NT

Inhibition of Sirtuins, Matrix Metalloproteinase, Bromodomains was Also Confirmed Using Standard Protocols and the Results are Present in Table 2.

Bioactivity of the above-described compounds has been verified by an anticancer cell proliferation assay using the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate) colorimetric assay by Roche Life Sciences®. Anticancer activity was tested against several standard cancer cell lines including brain, breast, Kaposi sarcoma, leukemia, lung, melanoma, tenosynovial giant cell tumor, ovarian, pancreatic, colon and prostate cancer. Cells were trypsinized and plated into 96 well plates in 50 μl of media and incubated overnight. The next day approximately 18 hours after plating, 50 μl of media containing the required flavonoid-based pharmaceutical composition was added per well. Cells were plated at a density so that 72 hours post drug addition, the cells will be in log phase (500)-2000 cells/well). The compounds and extracts were solubilized in Dimethyl sulfoxide (DMSO). The cells are allowed to proliferate for 72 hours 37° C. in humidified atmosphere of 5% CO₂. The experiment is terminated using WST-1 (Roche®) 10 μl per well and absorbance is read at 450 nm %690 nm. The effect of drugs on growth is assessed as percent of cell viability. The IC₅₀ values were determined from the extract dose versus control growth curves using Graphpad Prism® software. All experiments were carried out in duplicate and the mean results determined.

The results of the anticancer activity are presented in Tables 3 and 4 (above) and in FIG. 6, Hela cells shown at (A) and CMK cells at (B) and MV4-11 and MOLT-14 acute myeloid leukemia cells. To demonstrate a proof of concept in vivo, human pancreatic cancer xenograft CFPAC-1 cells implanted on scid mice were treated with FBL-03B and FBL-03G and demonstrated significant inhibition of tumor compared to the control. The results of the anti-pancreatic cancer activity in mice are presented in FIG. 7 and FIG. 8. To further demonstrate activity in-vivo, mice infected with acute myeloid leukemia cells harboring the FLT-3 mutation were treated with FBLGS-70 and its derivatives. The results of the anti-acute myeloid leukemia activity of FBLGS-70 and its derivatives are shown in FIG. 9.

It should now be apparent that the above-described invention provides a pharmaceutical composition for the prevention and treatment of disease with specific cannabis-based flavonoid compounds selected from among the groups of Cannflavin A, Canflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside and their derivatives selected from among the group of Geraldol, Rhamnetin, Isorhbamnetin, Rhamnazin, a method for the prevention and treatment of disease using the specific cannabis-based flavonoid pharmaceutical compositions, a method for isolating the cannabis-based flavonoid pharmaceutical compositions from raw plant material, and a method for synthesizing said specific cannabis-based flavonoid pharmaceutical compositions.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the claims. In addition, as one of ordinary skill in the art would appreciate, any dimensions shown in the drawings or described in the specification are merely exemplary, and can vary depending on the desired application of the invention. Many variations and modifications of the embodiments described herein will be obvious to one of ordinary skill in the art in light of the above disclosure. The scope of the invention is to be defined only by the claims, and by their equivalents.

STATEMENT OF INDUSTRIAL APPLICABILITY

Certain cancers overexpress certain oncogenic factors including kinases, sirtuins, bromodomains, matrix metalloproteinases and histone deacetylases, and so these particular oncogenic factors are useful therapeutic targets. Cannabis flavonoids have been shown to have several pharmacological properties against these oncogenic factors, but different cancers respond differently to oncogenic agents and that the response to a combination of several such agents is unpredicatable. There would be great industrial applicability in the use of a group of cannabis-based flavonoid pharmaceutical compositions selected from among the group of Cannflavin A, Cannflavin B, Cannflavin C, Chrysoeriol, Cosmosiin, Flavocannabiside and their derivatives selected from among the group of Geraldol, Rhamnetin, Isorhamnetin, Rhamnazin, for the prevention and treatment of certain cancers treatable by targeting kinases, sirtuins, bromodomains, matrix metalloproteinases and histone deacetylases. Some of the cancers that can be treated by use of cannabis flavonoids based on the inhibition of these therapeutic targets include brain, breast, colon, renal, liver, lung, lymphoma, pancreatic, pigmented villonodular synovitis prostate, leukemia, melanoma, tenosynovial giant cell tumor as well as any other cancers (e.g. solid, soft or hematological cancers) that overexpress the oncogenic factors inhibited by cannabis flavonoids and their derivatives. 

1. A cannabis-based flavonoid pharmaceutical composition for the prevention and treatment of cancer having a flavone backbone (2-phenyl-1,4-benzopyrone) chemical structure as shown below, or any pharmaceutically acceptable salt thereof:

wherein R1, R3, R5, R6, R7 and R10 may be none, any one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), a methyl group comprising one carbon atom bonded to three hydrogen atoms (CH3), an alkoxy group (O—CH3), a carboxyl group (COOH), chlorine (Cl), Bromine (Br), Fluorine (F), Glutamic acid (Glu), amine (NH2), amide (CONH2), imidazole (C3H4N2 and any salts or derivatives of the foregoing, R2, R4, R8 and R9 may be any one or more substituents selected from said group, and the carbon-to-carbon A and B bond is double flavone bond.
 2. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


3. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


4. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


5. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


6. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


7. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


8. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


9. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


10. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


11. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


12. The cannabis-based flavonoid pharmaceutical composition according to claim 1, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


13. A cannabis-based flavonoid pharmaceutical composition for the prevention and treatment of cancer and other diseases having a general flavone backbone (2-phenyl-1,4-benzopyrone) chemical structure as shown below, or any pharmaceutically acceptable salt thereof:

R2 may be one or more substituents selected from the group consisting of a hydrogen molecule (H), a hydroxide molecule (OH), or an oxygen molecule (O); R3 may be none, or one or more hydrogen molecules (H), R4 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or a nitrogen dioxide molecule (NO2); R5 and R8 may be none, or a hydroxide molecule (OH); R7 may be none, or the amine NH2; R9 may be none, or any one or more substituents selected from the group consisting of a hydroxide molecule (OH) or an oxygen molecule (O); and A and B are linked by a single bond.
 14. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


15. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


16. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


17. The cannabis-based flavonoid pharmaceutical composition according to claim 13, having a specific chemical structure as shown below, or any pharmaceutically acceptable salt thereof:


18. A method of treating cancer, the method comprising administering the general composition of claim
 1. 19. A method of treating cancer, the method comprising administering the specific composition of claim
 13. 20. A method of synthesizing the compound of claim 1, the method comprising: combining 4-coumaroyl-CoA with malonyl-CoA to yield a flavonoid backbone containing two phenyl rings; and promoting conjugate ring-closure of chalcones. 